Intercellular recognition complexes formed by T cell receptors (TCR) on cytotoxic T lymphocytes or T helper cells and MHC/peptide complexes on antigen presenting cells (APC) are a common recognition component in a diverse set of cell-cell encounters that activate T cells both during the development of the repertoire of T cells within an individual organism (positive selection; negative selection; peripheral survival) and during the control (T helper) and effector stages (T killer) of an adaptive immune response.
In the adaptive immune response, antigens are recognized by hypervariable molecules, such as antibodies or TCRs, which are expressed with sufficiently diverse structures to be able to recognize any antigen. Antibodies can bind to any part of the surface of an antigen. TCRs, however, are restricted to binding to short peptides bound to class I or class II molecules of the major histocompatibility complex (MHC) on the surface of APCs. TCR recognition of a peptide/MHC complex triggers activation (clonal expansion) of the T cell.
TCRs are heterodimers composed of two chains which can be αβ (alpha-beta) or γδ (gamma-delta). The structure of TCRs is very similar to that of immunoglobulins (Ig). Each chain has two extracellular domains, which are immunoglobulin folds. The amino-terminal domain is highly variable and called the variable (V) domain. The domain closest to the membrane is the constant (C) domain. These two domains are analogous to those of immunoglobulins, and resemble Fab fragments. The V domain of each chain has three complementarity determining regions (CDR). Proximal to the membrane, each TCR chain has a short connecting sequence with a cysteine residue that forms a disulfide bond between both chains.
Genes encoding αβ and γδ heterodimers are only expressed in the T-cell lineage. The four TCR loci (α, β, γ and δ) have a germ-line organization very similar to that of Ig. α and γ chains are produced by rearrangements of V and J segments whereas β and δ chains are produced by rearrangements of V, D, and J segments. The gene segments for TCR chains are located on different chromosomes, except the δ-chain gene segments that are between the V and J gene segments of the α chain. The location of δ-chain gene segments has a significance: a productive rearrangement of α-chain gene segments deletes C genes of the δ-chain, so that in a given cell the αβ heterodimer cannot be co-expressed with the γδ receptor.
In mice, there are about 100 Vα and 50 Jα genes segments and only one Cα segment. The δ-chain gene family has about 10 V, 2 D, and 2 J gene segments. The β-chain gene family has 20-30 V segments and two identical repeats containing 1 Dβ, 6 Jβ and 1 Cβ. Finally, the γ-chain gene family contains 7 V and 3 different J-C repeats. In humans the organization is similar to that of mice, but the number of segments varies.
The rearrangements of gene segments in α and β chains is similar to that of Igs. The α chain, like the light chain of Ig is encoded by V, J, and C gene segments. The β chain, like the heavy chain of Ig, is encoded by V, D, and J gene segments. Rearrangements of α chain gene segments result in VJ joining and rearrangements of β chain result in VDJ joining. After transcription of rearranged genes, RNA processing, and translation, the α and β chains are expressed linked by a disulfide bond in the membrane of T cells.
TCR gene segments are flanked by recognition signal sequences (RSS) containing a heptamer and a nonamer with an intervening sequence of either 12 nucleotides (one turn) or 23 nucleotides (two turn). As in Igs, enzymes encoded by recombination-activating genes (RAG-1 and RAG-2) are responsible for the recombination processes. RAG1/2 recognize the RSS and join V-J and V-D-J segments in the same manner as in Ig rearrangements. Briefly, these enzymes cut one DNA strand between the gene segment and the RSS and catalyze the formation of a hairpin in the coding sequence. The signal sequence is subsequently excised.
The combinatorial joining of V and J segments in α chains and V, D and J segments in β chains produces a large number of possible molecules, thereby creating a diversity of TCRs. Diversity is also achieved in TCRs by alternative joining of gene segments. In contrast to Ig, β and δ gene segments can be joined in alternative ways. RSS flanking gene segments in β and δ gene segments can generate VJ and VDJ in the β chain, and VJ, VDJ, and VDDJ on the δ chain. As in the case of Ig, diversity is also produced by variability in the joining of gene segments.
Hypervariable loops of the TCR known as complementarity determining regions (CDRs) recognize the composite surface made from a MHC molecule and a bound peptide. The CDR2 loops of α and β contact only the MHC molecule on the surface of APC, while the CDR1 and CDR3 loops contact both the peptide and MHC molecule. Compared with Ig, TCRs have more limited diversity in the CDR1 and CDR2. However, diversity of the CDR3 loops in TCRs is higher than that of Ig, because TCRs can join more than one D segment leading to augmented junctional diversity.
The pathogenesis of a number of autoimmune diseases is believed to be caused by autoimmune T cell responses to self-antigens present in the organism. Not all autoreactive T cells are deleted in the thymus, in contradiction with the clonal selection paradigm. Those T cells with TCRs for a broad spectrum of self-antigens represent part of the normal T-cell repertoire and naturally circulate in the periphery. It is unclear why autoreactive T cells are allowed, during their evolution, to undergo differentiation in the thymus and are accommodated in the periphery. While their physiological role is not understood, these autoreactive T cells, when activated, present a potential risk in the induction of autoimmune pathologies. Autoreactive T cells can also be isolated from normal individuals without the consequences of autoimmune diseases. It has been established that antigen recognition of autoreactivity by itself is not sufficient to mediate the autodestructive process. One of the prerequisites for autoreactive T cells to be pathogenic is that they must be activated.
Autoreactive T cells are implicated in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). The pathogenesis of autoreactive T cells in MS is generally held to arise from T cell responses to myelin antigens, in particular myelin basic protein (MBP). MBP-reactive T cells can be isolated from patients with MS but also from healthy individuals. One of the important discrepancies between myelin-reactive T cells in MS patients and those in normal individuals is the activation state of the T cells. MBP-reactive T cells are found to undergo in vivo activation, and occur at a higher precursor frequency in blood and cerebrospinal fluid in patients with MS as opposed to control individuals. These MBP-reactive T cells produce TH1 cytokines, e.g. IL-2, TNFα, and γ-interferon (IFNγ, which facilitate migration of inflammatory cells into the central nervous system and exacerbate myelin-destructive inflammatory responses in MS.
Microbial infections, in particular viral infections, may play an important role in the etiology and pathogenesis of multiple sclerosis (MS). It has been speculated that MBP-reactive T cells may be activated in vivo by viral infections in patients with MS through a mechanism of molecular mimicry, which has been supported by in vitro studies. A number of viruses, including measles virus, Epstein-Barr virus and most recently human herpesvirus-6 (HHV-6), have been implicated as etiologic agents in MS based on epidemiological evidence, geographic pattern and abnormal immune response to these viruses. The potential pathologic importance of certain viral infections in MS is thought to involve direct neurotropic properties of the virus, causing tissue damage, and their ability to activate autoimmune responses directed at myelin antigens, such as MBP, through the mechanism known as molecular mimicry.
Myelin-reactive T cells have been shown to be involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) in animals, which resembles MS. EAE can be induced actively in susceptible animals by injecting myelin proteins emulsified in an adjuvant or passively by injecting myelin-reactive T-cell lines and clones derived from myelin-sensitized animals. When activated in vitro, very small numbers of myelin-reactive T cells are required to induce EAE, while 100-fold more resting T cells with the same reactivity are incapable of mediating EAE.
EAE has been shown to be prevented and also treated by vaccination with inactivated myelin-reactive T cells, a procedure called T-cell vaccination (Ben-Nun et al., Nature, 1981; 292: 60-61). T-cell vaccination induces regulatory immune responses comprised of anti-idiotypic T cells and anti-ergotypic T cells, which lead to the depletion of myelin-reactive T cells. By depleting myelin-reactive T cells, therapeutic effects are observed in EAE and other experimental autoimmune disease models (Lider et al., Science, 1988; 239:820-822; Lohse et al. Science, 1989; 244: 820-822).
Due to the success in autoimmune disease models, T cell vaccination has recently advanced to clinical trials in patients with MS. Based on the results in experimental models such as EAE, it is believed that depletion of autoreactive T cells may improve the clinical course of MS and other autoimmune diseases.
In a pilot clinical trial, vaccination with irradiated autologous MBP-reactive T cell clones elicited CD8+ cytolytic T cell responses that specifically recognized and lysed circulating MBP-reactive T cells (Zhang et al., Science, 1993; 261: 1451-1454, Medaer et al. Lancet 1995: 346:807-808). Three subcutaneous inoculations with irradiated MBP-reactive T cell clones resulted in the depletion of circulating MBP-reactive T cells in patients with MS.
In a preliminary clinical trial, circulating MBP-reactive T cells were depleted in relapsing remitting MS patients and secondary progressive MS patients (Zhang et al., J Neurol., 2002; 249:212-8), by vaccinating the patients with three subcutaneous injections of irradiated autologous MBP-reactive T cells. T cell vaccination was beneficial to each group of patients as measured by rate of relapse, expanded disability scale score and MRI lesion activity. However, there was a trend for an accelerated progression after about twelve months following the last injection. The significance of the apparent accelerated progression is unknown, but may be associated with a gradual decline of the immunity induced by T cell vaccination against MBP-reactive T cells. In approximately 10-12% of the immunized patients, MBP-reactive T cells reappeared at about the same time as the accelerated progression. In some cases, the reappearing MBP-reactive T cells originated from different clonal populations that were not detected before vaccination, suggesting that MBP-reactive T cells may undergo clonal shift or epitope spreading. Clonal shift of MBP-reactive T cells has been observed in previous studies (Zhang et al. 1995) and may be associated with the on-going disease process.
Although T cell vaccination has been demonstrated to be effective for depleting myelin-reactive T cells and potentially beneficial for MS patients, there are several problems with the treatment. T cell vaccine treatment for each patient must be individualized because the T cell receptors of myelin-reactive T cells are highly diverse and vary between different MS patients (Vandevyver et al., Eur. J. Immunol., 1995; 25:958-968, Wucherpfennig et al., J. Immunol., 1994; 152:5581-5592, Hong et al., J. Immunol., 1999; 163:3530-3538).
In addition to being individualized for each patient, up to 8 weeks is required to produce a given T cell vaccine using current procedures. Production of a T cell vaccine begins with isolating mononuclear cells from the cerebrospinal fluid (CSFMCs) or peripheral blood (PBMCs) of a patient. The isolated mononuclear cells are then cultured with myelin peptides for 7-10 days to activate myelin-reactive T cells. Cultures are then tested for specific proliferation to myelin peptides by measuring [3H]-thymidine incorporation in the presence of myelin peptides over a period of 3 days. Cultures testing positive for specific proliferation to myelin peptides are then serially diluted to obtain clonal T cell lines or directly expanded. The cells are then cultured up to 6-8 weeks to expand the T cells. When the final T cell vaccine product is clonal, the T cells are homogenous with a single pattern of Vβ-Dβ-Jβ gene usage. Usually, three to six of the clonal cell lines are combined to produce a heterogeneous formulation with multiple patterns of Vβ-Dβ-Jβ gene usage. When the final T cell vaccine product is produced by direct expansion, the T cells are heterogeneous with more than one pattern of Vβ-Dβ-Jβ gene usage.
The individualized nature of T cell vaccination and the prolonged cell culture needed for production of each vaccine make treatment expensive and labor intensive under current methodologies. The extended time required for cell culture also creates a significant risk of contamination. Finally, the likelihood of clonal shift or epitope spreading of myelin-reactive T cells may require the subsequent production of a T cell vaccine for each patient with a different pattern of Vβ-Dβ-Jβ gene usage.
Therefore, there exists a need to develop improved methods of isolating T cells with specificity for antigens, such as MBP, that may be used to produce T cell vaccines for the treatment of patients with T cell-mediated diseases such as MS. There also exists a need to develop improved methods for producing T cell vaccines with a heterogeneous pattern of Vβ-Dβ-Jβ gene usage to account for clonal shift of autoreactive T cells.
In addition to vaccines based on inactivated T cells, vaccines may be based on immunogenic peptides. In EAE, encephalitogenic MBP-reactive T cells are restricted to very limited epitopes on MBP. These restrictions in the diversity of the pathogenic T-cell responses permit specific immune intervention. Various therapeutic strategies have been designed accordingly to target the Vβ region of the TCR in preventing the development of EAE in sensitized animals. For example, monoclonal antibodies have been targeted to the Vβ gene product and peptide vaccines have been based on the CDR2 region of the responsible Vβ gene.
Some of the studies on EAE have been extended to human autoimmune diseases. For instance, a peptide corresponding to TCR Vβ 5.2 has been used in clinical trials to treat patients with MS and a Vβ 14 peptide has been used to vaccinate patients with RA. U.S. Pat. No. 5,614,192 (Vandenbark) discloses treatment of autoimmune diseases by the use of immunogenic TCR peptides of 15 to 30 amino acids comprising at least part of CDR2. U.S. Pat. No. 6,303,314 (Zhang) discloses the treatment of autoimmune diseases by using certain immunogenic TCR peptides in combination with immunogenic T cell activation marker peptides.
One area in which vaccination with TCR peptides can be improved is by determining which, if any, common motifs are found in the autoreactive TCRs of a patient with an autoimmune disease such as MS. Such common motifs can be used either as a basis for a peptide vaccine to activate an anti-idiotypic immune response in MS patients for the purpose of depleting T cells which have TCRs comprising said motifs, or as a target for the preparation of antibodies that can functionally block or directly deplete T cells which have TCRs comprising said motifs.
Therefore, it is desirable to determine the amino acid sequences of common motifs specifically found in the TCRs of autoreactive T cells from patients with autoimmune diseases. It is also desirable to be able to readily detect such motifs in a patient sample by a convenient method, such as PCR. In addition, it is desirable to use peptides identical to or derived from the detected motifs to treat patients with the autoimmune disease. It is also desirable to use antibodies which specifically bind to said motifs to treat a patient with an autoimmune disease.
U.S. Pat. No. 6,303,314 (Zhang) discloses such a common motif found in the TCRs of a subset of Vβ13.1 T cells, the “LGRAGLTY motif”, which has the amino acid sequence Leu Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 10), as well as a method for its ready detection by PCR. This motif is found in some TCRs of some autoreactive T cells that recognize amino acids 83-99 of MBP (hereinafter “MBP83-99”). Peptides based on the LGRAGLTY motif can be used to vaccinate some patients in order to treat or prevent autoimmune diseases with which Vβ13.1-LGRAGLTY is associated (e.g., MS). U.S. Provisional Patent No. 60/386,287 (Zhang) discloses an additional three motifs present in the TCRs of MBP-reactive T cells.
As autoreactive T cells associated with autoimmune diseases, such as MS, may be activated by foreign antigens that mimic self-antigens, such as MBP, there remains a need to identify other TCR sequences, including CDR sequences, that cross-react with a self-antigen and a foreign antigen mimicking said self-antigen. In addition, there remains a need to be able to detect TCR sequences, including CDR sequences, which are expressed by MBP-reactive T cells that cross-react with a self-antigen and a foreign antigen mimicking said self-antigen. Finally, there remains a need to develop treatments for autoimmune diseases associated with TCR sequences, and more particularly CDR sequences, expressed by MBP-reactive T cells that cross-react with a self-antigen and a foreign antigen mimicking said self-antigen.